Successful embryo implantation requires correct development of the pre-implantation embryo, resulting in a hatched blastocyst which is able to implant into receptive endometrium. A considerable body of data has been collected which supports the idea that soluble growth factors, if secreted by the uterine epithelium, act directly on the embryo to control this process (Pampfer, S. et al, Bioessays, 13: 535-540 (1991); Tartakousky, B., and Ben Yair, E., Development Biology, 146: 345-352 (1991); Anderson, E. D., J. Cellular Biochem., 53: 280-287 (1993); and Schultz, G. A. and Hevner, S., Mutat. Res., 296: 17-31 (1992)).
In addition, developing embryos have been shown to produce a variety of cytokines which may act in an autocrine fashion on the endometrium to influence its receptivity. Examples of growth factors shown to be produced by human embryos include IL-1, IL-6, CSF-1 and TNF-.alpha. (Zolti et al, Fertil. Steril., 56 (1991) 265-272 and Witkin et al, J. Reprod. Immunol., 19 (1991) 85-93). TNF-.alpha. has been shown to be present in culture medium of human embryos up to the morula-st-age, but not that from the blastocyst (Lachappelle et al, Human Reproduction, 8: 1032-1038 (1993)). Production of cytokines by the embryo may therefore be regulated in a stage-specific manner.
Data on the possible direct effects of cytokines on embryos have come primarily from experiments in mice where many cytokines have been shown to affect the development of pre-implantation embryos in vitro. IFN-.gamma. and CSF-1, at physiological concentrations, inhibit the number of embryos developing to the blastocyst stage (Hill et al, J. Immunol., 139 (1987) 2250-2254). TNF-.alpha. has also been shown to have more subtle effects. Although TNF-.alpha. has no apparent effect on rates of blastocyst formation, it appears to specifically inhibit proliferation of cells contributing to the inner cell mass (ICM), which results in blastocysts with a reduced ICM (Pampfer et al, Endocrinology, 134: 206-212 (1994)).
Other growth factors also have specific effects on ICM cells. For instance, insulin-like growth factors 1 and 2 stimulate ICM proliferation, whereas leukaemia inhibitory factor (LIF) inhibits their differentiation (Harvey et al, Mol. Reprod. Dev., 31 (1992) 195-199).
It has been observed, in mouse systems, that embryos cultured in vitro lag in development compared to in vivo controls, and exhibit lower pregnancy rates after embryo transfer (Bowman, P. and McLaren, A., J. Embryol. Exp. Morphol., 24: 203-207 (1970)). Thus, a better understanding of the role of growth factors in development could lead to improved in vitro culture conditions and enhance the outcome in human IVF programs.
Stem cell factor (SCF) is a growth factor related in structure to CSF-1, and acts through the c-kit tyrosine kinase receptor. In bone marrow, SCF and CSF-1 act synergistically to promote proliferation and differentiation of stem cells into macrophage colonies.
EP-A-0423980 discloses the nucleic acid sequence of human SCF, and discusses potential uses of SCF in conditions requiring stimulation of cell proliferation, particularly blood cells.
In mouse, c-kit has been shown to be expressed throughout pre-implantation development (Arceci et al (1992)). We have now shown that the same is true in human embryos. At certain stages the human embryos also express SCF mRNA, suggesting that this growth factor may act in an autocrine fashion. This is in contrast to mouse, where no expression of SCF was detected in pre-implantation embryos (Arceci et al (1992)).
The full length SCF transcript consists of eight exons (Martin, F. H. et al, Cell, 63: 203-211 (1990)), which paper also discloses a variant form of SCF. A splice-variant of SCF has also been described which arises by virtue of the loss of exon 6 (Flanagan et al, Cell, 63: 1025-1035 (1991)).